Cell proliferation plays a critical role in the underlying events of human neoplasia, as well as our understanding of tumor biology as it relates to clinical course. The development of slide-based methods to assess cell proliferation is therefore of great interest with important potential applications. The overall goal of this project is to use monoclonal antibodies and cDNA probes to cell proliferation related markers (PCNA, H4 mRNA) and selected putative mediators of the loss of proliferation control [(p53, retinoblastoma gene product (RB1), the B chain of the platelet derived growth factor (PDGF-B)] on tissue sections of human soft tissue sarcomas and melanomas. A series of validation experiments will first be performed to permit the application of the cell proliferation markers to formalin and methacarn fixed, deparaffinized human tumor specimens, and to test the hypothesis that alterations in the distribution of proliferating cells within these tumors correlates with histologic progression to malignancy and/or predicts clinical outcome. It is then proposed to test the hypothesis that p53 overexpression, or loss of RB1 expression, correlates with alterations in tumor proliferation, as evidenced by quantitative and/or qualitative changes in the expression of PCNA or H4 mRNA, as determined by immuno- cytochemistry and in situ hybridization, in combination with cell-type specific markers. Quantification will be facilitated by employing image analysis with a microcomputer-based quantitative microscopy system. In addition, analysis of expression by human tumors of the tumor suppressor genes p53 and RB1, using immunocytochemistry, in situ hybridization, Western and Northern blots, will be performed to test the hypothesis that dysfunction of these proteins can define subsets of tumors with worse clinical outcomes. In addition, using these same tumors, the expression of PDGF-B chain, a growth factor, will be investigated using a series of monoclonal antibodies and cDNA probes in immunocytochemical as well as Northern blot analyses, to test the hypothesis that PDGF-B chain expression is correlated with paracrine stimulation of stromal cells in melanomas, and with autocrine stimulation in the case of soft tissue sarcomas. New monoclonal antibodies to p53, RB1, and PDGF-B chain protein which can be used on formalin fixed, paraffin embedded tissues will also be developed to facilitate retrospective analyses.